Method and apparatus for performing neuroimaging

ABSTRACT

The present invention relates to a restraining assembly used in neuroimaging of animals in magnetic resonance imaging (MRI) systems. The body of the animal under study is secured within a tube with a head holder to reduce motion artifacts, particularly when the animal is awake. The tube is placed in the bore of the MRI system to conduct imaging procedures with a radio frequency coil adjacent to the animal&#39;s head.

RELATED APPLICATION

[0001] This is a continuation of U.S. patent application Ser. No.09/169,602, filed Oct. 9, 1998 which is claims priority from U.S. patentapplication Ser. No. 09/073,546, filed May 6, 1998, the aboveapplications being incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to magnetic resonance imaging, andmore particularly to a method and apparatus for performing functionalmagnetic resonance imaging (fMRI) in animals.

BACKGROUND OF THE INVENTION

[0003] Human studies utilizing functional magnetic resonance imaging(fMRI) have advanced our understanding of the regional and functionalinterplay between populations of neurons serving sensory, integrativeand motor functions.

[0004] Changes in neuronal activity are accompanied by specific changesin hemodynamic functions such as cerebral blood flow, cerebral bloodvolume, and blood oxygenation. fMRI has been used to detect thesephysiologically induced changes in response to visual stimulation,somatosensory activation, motor tasks, and cognitive activity. Duringcognitive activity, the blood flow into the active region of the brainincreases considerably compared with the tissue oxygen uptake whichresults in an increase in blood oxy-hemoglobin (HbO₂) content. Thesusceptibility difference between diamagnetic oxy-hemoglobin andparamagnetic deoxy-hemoglobin (Hb) creates local magnetic fielddistortions that cause a dispersion in the processional frequency of thewater protons and a concomitant change in the magnetic resonance (MR)signal intensity which is proportional to the ration of HbO₂ to Hb.These signal-intensity alterations related to blood oxygenation aretermed the BOLD (blood oxygenation-level-dependent) effect. The voxelsin which paramagnetic Hb content is decreased are illuminated in theimage.

[0005] Unfortunately, extending these studies to animals has beendifficult because technological limitations prevent restraining aconscious animal for prolonged periods of time in a magnetic resonanceimaging (MRI) instrument. As a result most studies to date have beenlimited to animals which are typically anesthetized in order to minimizemotion artifacts. In the last 5 years over 7,000 fall lengthpublications on MRI in animals have been written without a singlereference to an awake animal. The low level of arousal during anesthesiaeither partially or completely suppresses the fMRI response and hasimpeded fMRI application to the more physiologically relevant functionsthat have been noted in humans.

[0006] Significant challenges remain in utilizing MRI techniques in bothhumans and anesthetized animals. One problem encountered in humanstudies has been artifacts from head movements. Studies in humans usinginvasive head fixation has shown improved image quality overnon-invasive fixation and absence of fixation. However, this fixationmethod limits the amount of research time available for human subjects.On the other hand, animal studies must be performed under anesthetizedconditions due to indiscriminate movement of conscious animals. Sinceimage resolution is a salient feature of fMRI, precautions to ensureimproved image quality with minimized head movements are essential. Inaddition to head movement, it has been observed that any motion outsidethe field of view can obscure or mimic the signal from neuronalactivation.

SUMMARY OF THE INVENTION

[0007] Applicant's method and apparatus overcomes the difficulties ofperforming fMRI on awake animals by utilizing a novel restrainingassembly to eliminate movement artifacts and to map neuronal activationafter exposure to sensorimotor stimulation in conscious animals. Thesignificance of applicant's method of neuroimaging in awake animals willchange current imagery of the brain from either a static (as seen withmost neurochemical measurements) or a low activation dynamic system inan anesthetized state (as seen with current fMRI or positron emissiontomography (PET) measurements) to a real-time three dimensionalfunctioning unit.

[0008] A novel stereotaxic assembly has been developed that canimmobilize the head and body of awake animals for several hours, withoutrestricting respiratory physiological functioning. The apparatus allowsfor collection of a consistent pixel by pixel representation of thebrain over several data acquisitions under various experimentalconditions. Applicants have demonstrated fMRI signal changes associatedwith neuronal activation in response to footshock and during odorstimulation. Changes are measured in conscious animals with and withoutthe use of contrast agents and are correlated with significantalterations in cerebral blood flow. Importantly, the information isobtained without animal sacrifice.

[0009] It has been found that the foregoing objects may be readilyobtained in the novel stereotaxic non-magnetic restraining assembly toimmobilize the head and body of awake animals for insertion into thetunnel bore of a magnetic resonance imaging assembly.

[0010] In a first embodiment of the invention, the assembly has agenerally planar horizontal chassis with a front mounting plate and rearmounting plate extending perpendicular to the chassis and locatedadjacent to each end of the chassis. A body tube bracket also extendsperpendicular to the chassis and is located between the front and rearmounting plates. The body tube bracket can be fastened (via aligningscrews) at different locations along the chassis to accept differentsized animals. The animal is placed in a body tube with its head in thecircular aperture of a head holder. The body tube slides into a centralaccess hole located in the approximate center of the rear mounting plateand the body tube bracket is thereby attached to the chassis. The headholder fastens to the chassis between the body tube bracket and thefront mounting plate.

[0011] The head holder restrains the head of the animal to prohibitvertical and horizontal movement of the animal during imaging. The headholder has a bite bar extending horizontally creating a chord along thebottom of its circular aperture. A vertical nose clamp extends throughthe top of the head holder and abuts the animal's nose to clamp theanimal's mouth thereon.

[0012] The animal's head is further restrained by a pair of lateral earclamping screws that extend horizontally through the sides of the headholder and a nose clamping screw that extends vertically through thehead holder. A protective earpiece is placed over the animal's ears andreceives the tips of the lateral ear clamping screws.

[0013] The head holder may be fitted with a radio frequency (rf) coilused to transmit rf radiation and receive the resulting MR signal.

[0014] A second embodiment of the invention has a general structuresimilar to the first embodiment with the following adaptations. The rearmounting plate has a removable crown to allow for simplified placementof the body tube into the rear mounting plate. In addition to the noseclamping screw as in the first embodiment, the means for restraining thehead includes two additional bottom jaw anchor screws located below thebite bar and extending radially inward toward the circular access holeto secure the animal's lower jaw against the horizontal bite bar. A headclamping screw extending located to the rear of the nose clamp andextending radially inward is included to further secure the animal'shead.

[0015] A further adaptation of the first embodiment includes a means ofrestraining an animal and prohibit limb movement. An animal is placedinto a restraining jacket that is wrapped at the back to restrain theanimal. Holders for the arms and legs further restrict the animal'smovement. Soft rubber ear pads may be fitted into the ear canals tominimize any irritation to the area and mollify background noise.

[0016] Accordingly, it is an object of this invention to provide a newand useful method and apparatus for performing neuroimaging on awakeanimals.

[0017] It is a further object of this invention to provide a method andapparatus for stereotaxically restraining an awake animal to preventmovement while undergoing fMRI.

[0018] Yet another object of this invention is to provide a stereotaxicrestraining assembly which is adaptable to different sized animals.

[0019] Another object of this invention is to amplify the sensitivity oflow field strength magnets with the use of exogenous contrast agents,blood oxygenation-level-dependent contrast and radio frequencysequences.

[0020] A further object of this invention is to register into athree-dimensional digital map of the brain created from a computerizedhistological representation of fMRI data obtained from fMRI scans.

[0021] Further objects and advantages of the present invention willbecome apparent from a consideration of the drawings and ensuingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] With respect to the first embodiment:

[0023]FIG. 1 is a side perspective of fMRI restraint assemblycomponents;

[0024]FIG. 2 is a side perspective view of the mounting unit;

[0025]FIG. 3 is a front view of the front mounting plate;

[0026]FIG. 4 is a front view of the rear mounting plate;

[0027]FIG. 5 is a front view of the body tube bracket;

[0028]FIG. 6 is a side perspective view of the body tube;

[0029]FIG. 7 is a front perspective view of the cylindrical head holder;

[0030]FIG. 8 is a side view of a rat in the cylindrical head holder;

[0031]FIG. 9 is a front view of a rat in the cylindrical head holder;

[0032]FIG. 10 is a front perspective view of a rat with thesemi-circular earpiece;

[0033]FIG. 11 is a side view of a rat with the semi-circular ear piece;and

[0034]FIG. 12 is a side view of a rat in the assembled fMRI restraint.

[0035] With respect to the second embodiment:

[0036]FIG. 13 is a side perspective view of the fMRI restraint assemblycomponents;

[0037]FIG. 14 is a view of the restraining jacket.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

[0038] To test the first embodiment of the invention, gold platedsurface electrodes were attached to the skin of the right or lefthindpaw of five male Sprague-Dawley rats (300-350 g) and connected to anelectrical stimulator that provided 25 V pulses of 0.3 ms duration of 3Hz (current of approximately 2.6 mA depending on skin resistance).Animals were lightly anesthetized with intraperitoneal administration ofchloral hydrate (300 mg/kg; Sigma, St. Louis, Mo.). The head was mountedand secured in a head holder custom fitted with a “birdcage” rf coil.The body of the animal was placed tightly into an animal holder,designed to allow for unrestricted respiration with lateral movement.Animals routinely recovered from the anesthesia within 30 mins., asevidenced by tail withdrawal, hindlimb movement and occasionalvocalizations. Blood pressure and heart rate were continuously monitoredfor signs of distress. The apparatus was inserted into the tunnel boreof a MRI device.

[0039] Magnetic resonance images were acquired using CSI-II 2.OT/45 cmimaging spectrometer (GE NMR Instruments, Fremont, Calif.) equipped withself-shielded gradient coils capable of producing a maximum fieldstrength of ±20 G/cm. The image processing was performed off-line on acomputer workstation (100 MHz Iris Indigo R4000 Silicon Graphics, Inc.,Mountain View, Calif.) and analyzed on a Power MAC 610/66 using NIHimage software (version 1.54). Prior to acquisition of the fMR images, aseries of scout images were acquired with an eight-slice, echo-planarimaging (EPI) sequence (field-of-view), FOV=25.6×25.6 mm; 64×64 datamatrix; 65 ms image acquisition window, to determine the exact positionof the animal. Approximately 120 minutes after positioning the animal inthe magnet, axial T₂*-weighted, BOLD images of the rat brain (underresting and stimulated conditions) were acquired using a 2Dgradient-echo imaging sequence (repetition time, TR=200 ms; echo time,TE=20 ms; 2-mm slice thickness; 128×128 data matrix). After a baselinestudy, the stimulus was applied for two minutes at 25 V and 1 minute at10 V before data collection. The resulting stimulated and baselineimages were subtracted to reveal regions of activation. The region ofgreatest activation were on the contralateral (to stimulated hind-paw)somatosensory frontal and parietal cortices was integrated from thesubtraction image by pixels±2SD above the signal-to-noise threshold withcomputer-assisted tomography. The corresponding region of the baselineand stimulated datasets were demarcated and the relative signalintensity was calculated on a pixel-by-pixel basis.

[0040] BOLD-based signal intensity was correlated to hemodynamic changesthrough measurement of relative cerebral blood flow (rCBF) in the regionof interest. A T₂*-weighted, gradient-echo EPI sequence (TR=900 ms;TE=38 ms: 65 ms image acquisition window, number of signal averages,NEX=1) was used to acquire 25 images from the same slice which gave themaximum BOLD signal intensity changes. A bolus of contrast agentgadopentetate dimeglumine (0.15 ml) was administered after the seventhimage. The change in the T₂* rate, ΛR₂*(t)=was obtained from the changein signal intensity based on the following relationship:ΛR₂*(t)=1n[S(t)S₀]/TE (where S(t) is the signal intensity at the timet). The relative cerebral blood volume (rCBV) and mean transit time(MTT) were determined for each pixel by the integration of ΛR₂*(t) andan estimate of the first moment of t, respectively. The rCBF wasdetermined, on a pixel-by-pixel basis, from the ratio of rCBV to MTT andrCBF maps were calculated from the 25 images. Since the cerebralhemodynamic state in a non-activated brain is quite stable over time,the resting-state rCBF maps were subtracted from the stimulated-staterCBF maps to create a new functional map depicting local changes causedby the hind-paw stimulation. Then the baseline and stimulated rCBF mapswere anatomically correlated to BOLD-based images allowing fordelineation of boundaries between the activated and the non-activatedregions. The relative signal intensity was calculated, on apixel-by-pixel basis, from the selected region.

[0041] Functional MRI can be performed in conscious animals providedthat there is adequate restraint. The acquired images had minimal motionartifacts, even with maximum stimulus strength. The signal enhancementwas related to stimulation intensity and was independent of whichhind-paw was stimulated. For example, the increase in signal intensitywith the 25 V stimulation was approximately 18% and with 100 V itincreased to 30%. The activated regions can be clearly discerned in thesubtraction images. The region of activation in individual animalsvaried with exact location and size since non-invasive skin electrodes(with large surface area) were used to minimize distress in theconscious animals.

[0042] Concurrent perfusion studies in the region identified by the BOLDtechnique showed corresponding increases in cerebral blood flow. Anaverage local increase in rCBV of 67±15% and in rCBF of 64±8%,corresponding to the initial BOLD changes of 18±1% (mean±SE, n−5), wasobserved. The subtraction images revealed good regional associationbetween the activated cortical region measured for rCBF and BOLD signalchanges, respectively. The average 1/T₂*, change of XXXX correspondenceto BOLD change of XXXX (mean±SE, n−X) was measured in the region ofinterest.

[0043] Since this study was done on conscious animals, comparisons withsimilar studies are somewhat limited. However, the results of this studyare consistent with a number of previous investigations. First, studiesexamining signal intensities in anesthetized animals post-stimulationrange from 5% obtained at 2.0 Tesla (T) to 5-17% (peak voxels 30%)obtained at 7.0T magnet. The relatively large BOLD signal intensitychanges observed in this study may be due to the increased neuronalactivation status of conscious animals, compared to the anesthetizedcounterparts. In this case, however, comparisons of signal intensitychanges between different studies can be misleading due to differencesin image acquisition parameters and/or magnetic field strengths. Tocircumvent this problem, applicant has attempted to estimate thesignal-intensity changes for our experimental conditions. In an in vivostudy, Prielmeier et al. Have determined 1/T₂* rates of rat brain duringhypoxia and interleaved normoxic phases (x). They found that 1/T₂*increases in a linear manner with arterial deoxygenation. In the study,a mild and moderate deoxygenation (less than 40%) corresponds to anapproximate change of 5.5 1/s in 1/T₂*. A severe deoxygenation (over40%) has lead to a plateau, which authors expect to result from enhancedcerebral blood flow (X). In applicant's study, a change of XXX in 1/T₂*was measured. Also, in an in-vitro study by Thulburn et al. (K. R.Thulbum, J. C. Waterton, P. M. Metthews, G. K. Radda, Biochemica etBiophysica Acta, 714, p. 265-270, 1982), a 1/T2 change of 8.3 S⁻¹correlated with a 75% change in oxygenation at 1.9T (close to our 2.0Tfield strength). Third, in a static mathematical model, an approximate60% change in blood oxygenation corresponded to a 64% increase in CBF at1.50 level of CBV. Taken together, the above studies support the currentquantitative data generated in conscious animals lining changes in BOLDand rCBF. Although one must be aware that extrapolation acrossdifferences in neuronal activation state (awake vs. anesthetized) andexperimental parameters, as well as interspecies variations, maycomplicate more direct comparisons. Furthermore, other reported stimulusduration and intensity dependent phenomena like signal saturation andundershoot after stimulation period were observed in these studies.

EXAMPLE II

[0044] To test a second embodiment of the invention, an adult malemarmoset was lightly anesthetized and fit into a custom-made clothjacket to keep the arms and legs from being pulled forward. A plasticsemicircular headpiece with blunted ear supports and soft rubber earpads were fit into the ear canals to minimize any irritation to the areaand mollify the sound of the radio frequency pulses. The marmoset's headwas placed into the cylindrical head restrainer with the animals caninessecured over a bite bar and ears positioned stereotaxically inside thehead restrainer with adjustable screws fit into lateral slits. The headholder was secured to the mounting unit with plastic screws. The body ofthe animal was placed into the body restrainer. The body restrainer wassecured onto the mounting unit and the assembly was placed into thetunnel bore of a MRI device. (The marmoset generally awoke fromanesthesia after approximately 45 to 60 minutes).

[0045] Magnetic resonance images were acquired on a General ElectricCSI-II 2.0-T/45-cm bore imaging spectrometer equipped with self-shieldedgradient coils capable of producing a maximum field strength of ±20 G/cm(General Electric Co., Fremont, Calif.). Prior to the experiment, thehead restrainer is custom-fit with a birdcage radio frequency coil.These coils are used to transmit the rf radiation and receive theresulting MR signal. Radio frequency coils are usually custom-fitted tothe desired anatomy to give maximum filling factor which results inoptimal sensitivity. A prototype birdcage coil of 5.8 cm diameter by 4.4cm in length was custom-fitted around the head holder. The circuitry wastuned to 85.557 MHz frequency.

[0046] There is a paucity of MRI data in non-human primates, hence itwas necessary to collect a set of serial images focusing on the brainanatomy of the adult male marmoset. T₁-weighted anatomical images wereacquired from three orthogonal planes using multi-slice spin-echoimaging sequence. Two sets of eight slices were acquired in aninterleaved fashion, resulting in 16 continuous slices, each 2 mm thick.The repetition time (TR) of acquisitions (NA) was 2 and the digitalresolution was 256×128.

[0047] Prior to acquisition of the fMR images, scout images from thethree planes were acquired with a single slice, spin-echo sequence(FOV=50×50 cm, digital resolution of 256×128, NA=2), to determine theexact position of the animal's head. BOLD based fMRI data sets wereacquired from rest, control and stimulated conditions using multi-slicegradient-echo sequence (TR=240 ms, TE=20 ms, NA=2, and digitalresolution of 128×128). First, a series of baseline images were acquiredto record background noise level and detect possible motion artifacts.

[0048] After baseline acquisitions were taken, data from the rest periodfor room air, stimulus scent (odor of a receptive female) and controlscent were collected. At the onset of olfactory stimulation, a stimuluscup was opened and placed 1.2 cm from the nose of the marmoset. A fanwas positioned at the back of the magnet pointing outward, pulling agentle draft of air through the bore. After three minutes, the stimuluscup was removed, exposing the animal to room air for two minutesfollowed by a three minute period with the control cup. This sequencewas repeated four times with a five minute rest period in between. Datawas collected during stimulus and control exposures. When the stimuluscup was not in use, it was sealed. Two sets of images were acquiredduring each presentation of the scent.

[0049] The image processing was performed off-line on a 100 MHz HPApollo 735 workstation using IDL imaging software, Version 4.0 andanalyzed on a Power Mac 60/66 using NIH imaging software, Version 1.56(Apple Computer, Inc., Cupertino, Calif.). The stimulated and baselineimages were subtracted to reveal regions of activation. The region ofgreatest activation was determined from the subtraction image. Thecorresponding region of the baseline and stimulated data sets weredemarcated and the relative signal intensity was calculated on apixel-by-pixel basis. Brain activity increased with time of exposure tothe scent of the receptive female.

DETAILED DESCRIPTION OF THE DRAWINGS

[0050] Turning first to FIG. 1, therein illustrated is a disassembledfMRI restraining assembly having a Plexiglas™ chassis generallydesignated by the numeral 1, a Plexiglas™ cylindrical head holdergenerally designated by the numeral 2, a Plexiglas™ body tube generallydesignated by the numeral 3, a rear mounting plate generally designatedby numeral 7, a front mounting plate generally designated by numeral 8,and a body tube bracket generally designated by numeral 9. One end ofthe chassis 1 is fitted into chassis mounting slot 10 (not shown) ofrear mounting plate 7 and the opposite end of chassis 1 is fitted intochassis mounting slot 10 of the front mounting plate 8.

[0051] Turning in detail to the assembly as seen in FIG. 2, therein isillustrated an elongated rectangular Plexiglas™ chassis 1 with a seriesof parallel opposing adjusting holes 6 drilled approximately midwaytherein. At one end of the chassis 1 is a circular Plexiglas™ rearmounting plate 7 and at the opposite end of the chassis is a circularPlexiglas™ front mounting plate 8. Also, shown in FIG. 2 is a squarePlexiglas™ body tube bracket 9 adjustably mounted to the chassis 1 by apair of screws 35 (shown in FIG. 13) extending through the chassis inthe correlating mounting screw holes 6 and into body bracket tube 9.

[0052]FIG. 3 is a detailed view of the front mounting plate 8 having acentrally located circular access hole 11 extending through theapproximate mid-section of the front mounting plate and a chassismounting slot 10 extending horizontally below the circular access hole11 which receives the chassis 1. Screw alignment slots 12 are locatedradially from the central circular access hole 11 to allow accessthrough the front mounting plate 8 for adjustment of head holder 2. Anassembly mounting block 13 and assembly mounting screw 14 penetratingthe assembly mounting block 13 are located an outer surface of the frontmounting plate 8 above the circular access hole 11 to secure theassembly in the cylindrical bore of a MRI tunnel (not shown). Atradially equidistant points located on the perimeter of the frontmounting plate 8 are three assembly centralizers 15 which providefurther stability to the assembly when placed into the cylindrical boreof a MRI tunnel.

[0053]FIG. 4 shows a detailed view of the rear mounting plate 7 having acentral circular access hole 11 and a horizontal chassis mounting slot10 extending horizontally below the circular access hole 11 whichreceives the chassis 1. Arcuate cable access slot 16 is located abovethe access hole 11 to allow access through the rear mounting plate 7. Atradially equidistant points located on the perimeter of the rearmounting plate are three assembly centralizers 15 provide furtherstability to the assembly when placed into the cylindrical bore of a MRItunnel. Assembly centralizers 15 also act as feet to stabilize theassembly when it is free standing outside the cylindrical bore of a MRItunnel.

[0054]FIG. 5 shows a detail of the body tube bracket 9 which has anaccess hole 11 therein for receiving the body tube 3 and a clampingscrew hole 17 located through the top surface to receive a clampingscrew 18 for temporarily fastening the body tube 3 to the body tubebracket 9. The bottom surface of the body tube bracket 9 has a pair ofthreaded mounting screw holes 19 for receiving aligning screws 35 (shownin FIG. 13) which detachably attach the body tube bracket 9 to thechassis 1.

[0055]FIG. 6 shows the body tube 3 which is an elongated Plexiglas™ tubehaving two elongated animal access slots 20. By turning to FIG. 12 itwill be appreciated that body tube 3 may be slideably inserted throughaccess hole 11 of the rear mounting plate 7 and into access hole 11 ofbody tube bracket 9. Once inserted, clamping screw 18 may be tightenedto releasably secure the body tube 3 in body tube bracket 9.

[0056]FIGS. 7, 8 and 9 illustrate the head holder 2 having lateral earclamping screws 4 inserted into lateral screw slots 21. The head holder2 is a cylindrical tube having a central aperture 22 therethrough forreceiving the head of an animal 23 and a bite bar 24 extendinghorizontally along a chord of the circular aperture 22 to provide a restfor the upper jaw of a restrained animal 23. Mounted through the top ofthe cylindrical head holder is a nose clamping screw 25 to secure thenose of a restrained animal 23 to the bite bar 24 as shown in FIG. 9. Apair of opposed lateral screw slots 21 are located in the sides of thecylindrical head holder to receive lateral ear clamping screws 4.Encompassing the head holder 2 is a birdcage coil 26. The head holder 2is connected to the chassis 1 by a pair of mounting screws 29 (notshown) extending through the chassis 1 and into the head holder mount28.

[0057] As shown in FIGS. 10 and 11, the semi-circular earpierce 5 isfitted over the head of the animal 23 whereupon the animal's head isplaced into head holder 2. Lateral ear clamping screws 4 are insertedthrough a pair of lateral screw slots 21 and tightened against divots ina semi-circular earpiece 5 to prevent the animal from movinghorizontally. The upper jaw of the animal 23 is fitted over the bite bar24 and nose clamping screw 25 is tightened against the snout of theanimal to secure it to the bite bar 24 and thereby eliminate verticalmovement maintaining a stereotaxic position of the animal's head.

[0058] As shown in FIG. 12, the cylindrical head holder is fixedlymounted to the chassis 1 by a pair of head holder mounting screws 29threadably fastened into the head holder mount 28 allow adjustment ofthe cylindrical head holder 2 to properly fit different size animals.Once assembled the various components of the fMRI restraining assemblycooperate to minimize movement in an awake animal and thereby allows fora method of performing neuroimaging on an awake animal 23.

[0059] Turning now to FIG. 13 depicting a side perspective view of asecond embodiment of the restraining assembly having an elongatedrectangular chassis designated by the numeral 1, a Plexiglas™ headholder usually designated by the numeral 2, a Plexiglas™ body tubegenerally designated by the numeral 3, a rear mounting plate generallydesignated by the numeral 7, a front mounting plate generally designatedby the numeral 8, and a body tube generally designated by the numeral 8,and a body tube generally designated bracket by the numeral 9. The frontmounting plate 8 has an access hole extending horizontally therethrough.Attached to the top front of the front mounting plate 8 is an assemblymounting block 13 and screw 14 and attached to the bottom front of thefront mounting pate 8 is an anchor screw 30. The assembly mounting block13 and the front anchor screw 30 are adapted to hold the assembly inplace when inserted into the magnetic bore of a MRI tunnel. The frontmounting plate 8 and rear mounting plate 7 each contain a chassismounting slot 10 along the interior bottom to accept and interlock withthe chassis 1.

[0060] A series of parallel opposing adjusting holes 6 are locatedapproximately in the middle section of chassis 1. The head holder 2connects to the chassis 1 through head holder mounting screw 29 andcorresponding adjusting holes 6. The head holder 2 has a centralgenerally circular aperture running horizontally therethrough and alongthe central axis of the chassis. The head holder 2 has a head clampingscrew 31 located at the top front of the head holder 2 extendingradially downward to the front of the head holder 2 and extends into thecentral aperture. The head holder 2 also has a nose clamping screw 25 atthe top front of the head holder 2 extending radially downward to therear of the head holder 2 and extends into the central aperture. Thehead holder 2 further has a pair of jaw screws 32 located at the bottomfront of the head holder 2 and extending radially inward into the headholder's central aperture.

[0061] Ear clamping screws 4 are located on opposite sides of the headholder 2 within the lateral screw slots 21 and extend horizontally intothe central axis of the chassis. The lateral screw slots 21 are locatedon opposite sides of the head holder 2 and extend from the rear edge ofthe head holder 2 toward the front of the head holder 2. The restrainedanimal wears a semi-circular earpiece 6 and soft rubber pads 36 toprotect the animal's ear canals. The animal's head is fitted into thehead holder 2 and the ear clamping screws 4, which slide into thelateral screw slots 21, fasten onto divots on the disk end portions ofthe semi-circular earpiece 6.

[0062] The body tube 3 is an elongated Plexisglas™ cylinder attached tothe chassis 1 through a rear mounting plate 7 at the end of the chassisopposite the front mounting plate 8. An anchor screw 30 is located atthe bottom rear of the rear mounting plate 7 that is adapted to hold theassembly in place when inserted into the magnetic bore of a MRI tunnel.The rear mounting plate 7 has an access hole running generallyhorizontally through the center to receive and align the body tube 3.The rear mounting plate 7 also contains a body tube clamp 33 located atthe top of the rear mounting plate 7. The rear mounting plate 7 has achassis mounting slot 10 along the bottom front to accept and interlockwith the chassis 1. The rear mounting plate 7 further has a removablecrown 34 to allow easier placement of the body tube 3 into rear mountingplate 7. The rear body tube clamp 33 acts to hold the crown in place.

[0063] The body tube bracket 9 is located along the chassis 1 betweenthe head holder 2 and the rear mounting plate 7 and is adjustablyattached to the chassis 1 by a pair of aligning screws 35 (not shown) ina corresponding adjusting holes 6. The body tube bracket 9 has an accesshole running horizontally therethrough to receive and align the bodytube 3. A clamping screw 18 and corresponding hole are located at thetop of the body tube bracket 9 pointing downwards to holds the body tubein place.

[0064]FIG. 14 describes a novel restraining jacket 37 used to restrainan animal. The jacket is made of a Velcro™ lined, non-flexible fabricwith a Velcro™ closure 38. Arm and leg holders 39 and 40, respectively,further restrict the animal's movement. The jacket has holes for theanimal's head 41 and 42, respectively.

[0065] The present invention demonstrates novel images of neuronalactivation in conscious animals. Current methods utilizing anesthetizedanimals, which are known to exhibit dampened neuronal activity, may masklow signals levels. Furthermore, since the level of arousal (consciousvs. anesthetized) is inextricably linked to behavior, the future use ofthis assembly will be a significant step in providing a betterunderstanding of the neural circuitry that facilitates behaviors such asresponses to visual stimulation, temperature regulation, and motorstimulation, in addition to a range of different environmental stressorsand interneurodevelopmental and intraneurodevelopmental studies.Therefore, researchers interested in the brain and/or behavior(utilizing laboratory animals) will be further assisted in theirdiscoveries, with the utilization of this new assembly.

[0066] It will be appreciated that the above description contains manyspecificities, these should not be construed as limitations on the scopeof the invention, but rather as an exemplification of a preferredembodiment thereof. Many other variations are possible.

1. A restraining assembly to immobilize an awake animal for magneticresonance imaging (MRI) device, comprising: a chassis; a first mountingplate at one end of the chassis; a second mounting plate at a second endof said chassis opposite said first mounting plate; an elongated bodytube extending along said chassis; and a head holder that restrains thehead of an animal, the head holder having an rf coil.
 2. The restrainingassembly of claim 1 wherein said head holder further comprises a pair oflateral ear clamping screws extending horizontally through the sides ofsaid head holder into the bore of said aperture and generallyperpendicular to an elongated axis thereof and above a horizontal bitebar, and a protective ear piece.
 3. The restraining assembly of claim 1wherein said head holder further comprises a nose clamping screwextending inward through the top of said head holder into a bore of saidaperture.
 4. The restraining assembly claim 1 wherein said head holderfurther comprises a pair of jaw anchor screws extending inward throughsaid head holder into bore of said aperture.
 5. The restraining assemblyclaim 1 wherein said head holder further comprises a head clamping screwlocated at the top of said head holder and extending inward through saidhead holder into a bore of said aperture.
 6. The restraining assembly ofclaim 1 further comprising a restraining jacket for immobilizing theanimal.
 7. The restraining assembly claim 2 further comprising ear padswherein said ear pads are placed under said protective ear piece.
 8. Amethod of imaging a brain of a conscious awake animal, comprising:restraining an un-anesthetized animal in an assembly slidably mounted inan MRI device, the assembly including a chassis; a front mounting platelocated at a first end of the chassis; a rear mounting plate located ata second end of the chassis opposite the front mounting plate; anelongated body tube extending along the chassis the tube enclosing theanimal; and conducting an imaging procedure on the brain consciousanimal.
 9. The method of claim 8 further comprising the step ofamplifying the sensitivity of low field strength magnets by implementingexogenous contrast agents, blood oxygenation-level-dependant contrastand radio frequency sequences.
 10. The method of claim 8 furthercomprising the steps of: obtaining a computerized histologicalrepresentation of an fMRI signal; and forming a three dimensionaldigital map of the imaged brain from a computerized histologicalrepresentation of the fMRI signal
 11. The method of claim 9 furthercomprising the steps of: obtaining a computerized histologicalrepresentation of a fMRI signal; and forming a three dimensional digitalmap of the imaged brain from a computerized histological representationof the fMRI signal.
 12. The method of claim 8 further comprisingperforming an fMRI sequence using a real-time three dimensionalfunctioning unit.
 13. The method of claim 9 further comprisingperforming an fMRI sequence using a real-time three dimensionalfunctioning unit.
 14. Apparatus for restraining a conscious animalduring a magnetic resonance imaging procedure comprising; a head holderwith which an animal's head is restrained; a chassis mounted within anMRI device on which the head holder is attached, the chassis having afirst end plate and a second end plate mounted thereon such that theanimal's body is positioned between the first end plate and the secondend plate; and a body tube in which the animal's body is enclosed. 15.The apparatus of claim 14 further comprising an rf coil that is mountedon the head holder.
 16. The apparatus of claim 14 further comprising ahole in the second end plate, the body tube having a size such that thebody tube can be inserted through the hole.
 17. The apparatus of claim14 further comprising a clamp on the head holder that is be secured tothe animal's head and a bite bar.
 18. The apparatus of claim 14 furthercomprising a restraining jacket that restrains an un-anesthetized animalduring an imaging procedure.